One reason why pancreatic cancer is so aggressive and unresponsive to treatments is its resistance to apoptosis. We report here that reactive oxygen species (ROS) are a prosurvival, antiapoptotic factor in pancreatic cancer cells. Human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cells generated ROS, which was stimulated by growth factors (serum, insulin-like growth factor I, or fibroblast growth factor-2). Growth factors also stimulated membrane NAD(P)H oxidase activity in these cells. Both intracellular ROS and NAD(P)H oxidase activity were inhibited by antioxidants tiron and N-acetylcysteine and the inhibitor of flavoprotein-dependent oxidases, diphenylene iodonium, but not by inhibitors of various other ROS-generating enzymes. Using Rho⁰ cells deficient in mitochondrial DNA, we showed that a nonmitochondrial NAD(P)H oxidase is a major source of growth factor-induced ROS in pancreatic cancer cells. Among proteins that have been implicated in NAD(P)H oxidase activity, MIA PaCa-2 and PANC-1 cells do not express the phagocytic gp91^phox subunit but express several nonphagocytic oxidase (NOX) isoforms. Transfection with Nox4 antisense oligonucleotide inhibited NAD(P)H oxidase activity and ROS production in MIA PaCa-2 and PANC-1 cells. Inhibiting ROS with the antioxidants, Nox4 antisense, or MnSOD overexpression all stimulated apoptosis in pancreatic cancer cells as measured by internucleosomal DNA fragmentation, phosphatidylserine externalization, cytochrome c release, and effector caspase activation. The results show that growth factor-induced ROS produced by NAD(P)H oxidase (probably Nox4) protect pancreatic cancer cells from apoptosis. This mechanism may play an important role in pancreatic cancer resistance to treatment and thus represent a novel therapeutic target.

Background & Aims Cigarette smoke has been identified as an independent risk factor for chronic pancreatitis (CP). Little is known about the mechanisms by which smoking promotes development of CP. We assessed the effects of aryl hydrocarbon receptor (AhR) ligands found in cigarette smoke on immune cell activation in humans and pancreatic fibrosis in animal models of CP. Methods We obtained serum samples from patients with CP treated at Stanford University hospital and healthy individuals (controls) and isolated CD4⁺ T cells. Levels of interleukin-22 (IL22) were measured by enzyme-linked immunosorbent assay and smoking histories were collected. T cells from healthy nonsmokers and smokers were stimulated and incubated with AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene) or antagonists and analyzed by flow cytometry. Mice were given intraperitoneal injections of caerulein or saline, with or without lipopolysaccharide, to induce CP. Some mice were given intraperitoneal injections of AhR agonists at the start of caerulein injection, with or without an antibody against IL22 (anti-IL22) starting 2 weeks after the first caerulein injection, or recombinant mouse IL22 or vehicle (control) intraperitoneally 4 weeks after the first caerulein injection. Mice were exposed to normal air or cigarette smoke for 6 h/d for 7 weeks and expression of AhR gene targets was measured. Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. Pancreatic stellate cells and T cells were isolated and studied using immunoblot, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent analyses. Results Mice given AhR agonists developed more severe pancreatic fibrosis (based on decreased pancreas size, histology, and increased expression of fibrosis-associated genes) than mice not given agonists after caerulein injection. In mice given saline instead of caerulein, AhR ligands did not induce fibrosis. Pancreatic T cells from mice given AhR agonists and caerulein were activated and expressed IL22, but not IL17 or interferon gamma. Human T cells exposed to AhR agonists up-regulated expression of IL22. In mice given anti-IL22, pancreatic fibrosis did not progress, whereas mice given recombinant IL22 had a smaller pancreas and increased fibrosis. Pancreatic stellate cells isolated from mouse and human pancreata expressed the IL22 receptor IL22RA1. Incubation of the pancreatic stellate cells with IL22 induced their expression of the extracellular matrix genes fibronectin 1 and collagen type I α1 chain, but not α2 smooth muscle actin or transforming growth factor−β. Serum samples from smokers had significantly higher levels of IL22 than those from nonsmokers. Conclusions AhR ligands found in cigarette smoke increase the severity of pancreatic fibrosis in mouse models of pancreatitis via up-regulation of IL22. This pathway might be targeted for treatment of CP and serve as a biomarker of disease.

The extracellular matrix (ECM) facilitates pancreatic cancer cells survival, which is of central importance for pancreatic adenocarcinoma that is highly fibrotic. Here, we show that reactive oxygen species (ROS) mediate the prosurvival effect of ECM in human pancreatic cancer cells. Fibronectin and laminin stimulated ROS production and NADPH oxidase activation in pancreatic cancer cells. Both pharmacological and molecular approaches show that fibronectin stimulated ROS production through activation of NADPH oxidase and NADPH oxidase-independent pathways and that 5-lipoxygenase (5-LO) mediates both these pathways. Analyses of the mechanisms of ROS production by ECM proteins and growth factors indicate that activation of NADPH oxidase (Nox4) is a common mechanism employed both by ECM proteins and growth factors to increase ROS in pancreatic cancer cells. We also found that Nox4 is present in human pancreatic adenocarcinoma tissues and that these tissues display membrane NADPH oxidase activity. ECM proteins and growth factors activate NADPH oxidase through different mechanisms; in contrast to ECM proteins, growth factors activate NADPH oxidase through 5-LO-independent mechanisms. Inhibition of 5-LO or NADPH oxidase with pharmacological inhibitors of these enzymes and with Nox4 or 5-LO antisense oligonucleotides markedly stimulated apoptosis in cancer cells cultured on fibronectin. Our results indicate that ROS generation via 5-LO and downstream NADPH oxidase mediates the prosurvival effect of ECM in pancreatic cancer cells. These mechanisms may play an important role in pancreatic cancer resistance to treatments and thus represent novel therapeutic targets.